Lab Exercise 11 - Gram-Negative Rods

Enterobacteriaceae

a family of Gram-negative rods that all ferment glucose, test nitrate positive, and oxidase negative

most are motile

intestinal pathogens

many symbionts, many potential pathogens

the complete Enterobacteriaceae dichotomous key

lactose test + → IMVIC test

lactose test - → H2S motility test

IMViC: I+, M+, V-, C- → Escherichia coli

IMViC: I-, M-, V+, C+ → Enterobacter aerogenes

H2S motility test + → Shigella sonnei

H2S motility test - → urea test

urea test + → Proteus mirabilis

urea test - → Salmonella spp.

what is EMB selective and differential for?

selective for Gram-negatives

differential for lactose fermentation

What is MacConkey agar selective and differential for?

selective for Gram-negatives

differential for lactose fermentation

What is SS agar selective and differential for?

selective for Gram-negatives

differential for lactose fermentation and sulfur reduction

What is BSA selective and differential for?

selective for Salmonella spp.

differential for sulfur reduction

positive result of MacConkey agar, and what it indicates

growth with pink color, indicates Gram-negative with lactose fermentation

negative result of MacConkey agar, and what it indicates

growth without pink color, indicates Gram-negative without lactose fermentation

strong positive result for EMB, and what it indicates

growth with dark purple color or a metallic green sheen, indicates Gram-negative with strong lactose fermentation

weak positive result for EMB, and what it indicates

growth with pink/lavender growth, indicates Gram-negative with weak lactose fermentation

negative result of EMB, and what it indicates

growth without a color change, indicates Gram-negative without lactose fermentation

lactose-positive result for SS, and what it indicates

growth with pink to red color

indicates Gram-negative with lactose fermentation and no sulfur reduction

sulfur-positive result for SS, and what it indicates

growth with black precipitate

indicates Gram-negative with sulfur reduction and no lactose fermentation

negative result for SS, and what it indicates

growth without any color change or precipitate, indicates Gram-negative without lactose fermentation or sulfur reuction

positive result for BSA, and what it indicates

growth with black precipitate, indicates Gram-negative with sulfur reduction

negative result for BSA, and what it indicates

growth without precipitate, indicates Gram-negative without sulfur reduction

positive result for tryptone broth test, and what it indicates

cerise ring at the top of sample, indicates tryptophanase → indole production and reaction with Kovac’s reagent

bacteria is E. coli

negative result for tryptone broth, and what it indicates

yellow ring (no cerise), indicates lack of tryptophanase → no indole

bacteria is E. aerogenes

positive result for MR, and what it indicates

reagent remains red, indicates strong acid production

bacteria is E. coli

negative result for MR, and what it indicates

red color is not retained, indicates weak acid production

bacteria is E. aerogenes

positive result for VP, and what it indicates

red color after addition of VP I and VP II, indicates 2,3-butanediol fermentation

bacteria is E. aerogenes

negative result for VP, and what it indicates

no red color after addition of Barritt’s reagents (remains yellow), indicates lack of fermentation

bacteria is E. coli

positive result for Simmons citrate, and what it indicates

growth, often with blue color, indicates citrate is used as the sole carbon source

bacteria is E. aerogenes

negative result for Simmons citrate, and what it indicates

no growth, color remains green → indicates citrate was not the sole carbon source

bacteria is E. coli

what is SIM used for?

to determine sulfur reduction, indole production, and motility

differentiates Shigella sonnei from Salmonella spp. and Proteus mirabilis

positive result for SIM, and what it indicates

black precipitate, cerise ring, and turbidity → indicate sulfur production, tryptophanase presence, and motility

bacteria could be Proteus mirabilis or Salmonella spp., but not Shigella sonnei

negative result for SIM, and what it indicates

no black precipitate, cerise ring, or turbidity → indicates no sulfur production, tryptophanase absence, and no motility

bacteria is Shigella sonnei

What is urea broth used for?

detects urease

differentiates Proteus mirabilis from Salmonella spp.

positive result for urea broth, and what it indicates

color change to cerise, indicates pH increase as urea degrades to NH3 and CO2

bacteria is Proteus mirabilis

negative result for urea broth, and what it indicates

color either stays red or changes to yellow, indicates pH remains neutral or decreases as acid is possibly produced

bacteria is Salmonella spp.